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Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids., Competing Interests: Declaration of competing interest Manchester Biogel in part funded this research through a PhD studentship to the lead author (EL). Aline Miller and Alberto Saiani were shareholders, directors, and consultants for Manchester Biogel., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Apoptosis is regulated by interactions between the BH3-only and multi-domain Bcl-2 family proteins. These interactions are integrated on the outer mitochondrial membrane (OMM) where they set the threshold for apoptosis, known as mitochondrial priming. However, how mitochondrial priming is controlled at the level of single cells remains unclear. Retrotranslocation of Bcl-XL has been proposed as one mechanism, removing pro-apoptotic Bcl-2 proteins from the OMM, thus reducing priming. Contrary to this view, we now show that Bcl-XL retrotranslocation is inhibited by binding to its BH3-only partners, resulting in accumulation of these protein complexes on mitochondria. We find that Bcl-XL retrotranslocation dynamics are tightly coupled to mitochondrial priming. Quantifying these dynamics indicates the heterogeneity in priming between cells within a population and predicts how they subsequently respond to a pro-apoptotic signal., (© 2022. The Author(s).)

Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming. Underlying this process, we reveal a key role for mitochondrial dynamics in the regulation of DNA damage and genome instability. The ability of mitochondrial dynamics to regulate oncogenic DNA damage centers upon the control of minority mitochondrial outer membrane permeabilization (MOMP), a process that enables non-lethal caspase activation leading to DNA damage. Mitochondrial fusion suppresses minority MOMP and its associated DNA damage by enabling homogeneous mitochondrial expression of anti-apoptotic BCL-2 proteins. Finally, we find that mitochondrial dysfunction inhibits pro-apoptotic BAX retrotranslocation, causing BAX mitochondrial localization and thereby promoting minority MOMP. Unexpectedly, these data reveal oncogenic effects of mitochondrial dysfunction that are mediated via mitochondrial dynamics and caspase-dependent DNA damage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong β-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes β-catenin. Deletion of this exon yields unconstrained β-catenin signalling activity. We also demonstrate that β-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which β-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways., (© 2021. The Author(s).)

Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how it is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to antimitotic drugs. Here, we report an unbiased proximity biotinylation (BioID) screen to identify regulators of apoptotic priming in mitosis, using Bid as bait. The screen primarily identified proteins outside of the canonical Bid interactome. Specifically, we found that voltage-dependent anion-selective channel protein 2 (VDAC2) was required for Bid phosphorylation-dependent changes in apoptotic priming during mitosis. These results highlight the importance of the wider Bcl-2 family interactome in regulating the temporal control of apoptotic priming.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo , regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity., (© 2020 Kuwana et al.)

Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured and reversible protein-level response, even as transcription is apparently downregulated. Protein abundance is quantified coincident with changes to protein conformation and post-translational modification (PTM). Furthermore, we characterize changes to the linker of nucleoskeleton and cytoskeleton (LINC) complex that bridges the nuclear envelope, and specifically to levels and PTMs of Sad1/UNC-84 (SUN) domain-containing protein 2 (SUN2). The result of this regulation is to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity., (© 2017 The Authors.)

Epithelial cells forming mammary gland ducts and alveoli require adhesion to the extracellular matrix for their function. Mammary epithelial cells need β1-integrins for normal cell cycle regulation. However, the role of β1-integrins in tumorigenesis has not been fully resolved. β1-integrin is necessary for tumour formation in transgenic mice expressing the Polyomavirus Middle T antigen, but it is dispensable in those overexpressing ErbB2. This suggests that some oncogenes can manage without β1-integrin to proliferate and form tumours, while others still require it. Here we have developed a model to test whether expression of an oncogene can surpass the need for β1-integrin to drive proliferation. We co-expressed the ErbB2 or Akt oncogenes with shRNA to target β1-integrin in mammary epithelial cells, and found that they show a differential dependence on β1-integrin for cell division. Moreover, we identified a key proliferative role of the Rac1-Pak axis downstream of β1-integrin signalling. Our data suggest that, in mammary epithelial cells, oncogenes with the ability to signal to Pak surpass the requirement of integrins for malignant transformation. This highlights the importance of using the correct combination therapy for breast cancer, depending on the oncogenes expressed in the tumour., (Copyright © 2017. Published by Elsevier GmbH.)

Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1-integrin (β1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of β1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in β1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

A key hallmark of cancer cells is the loss of positional control over growth and survival. Focal adhesion kinase (FAK) is a tyrosine kinase localised at sites of integrin-mediated cell adhesion to the extracellular matrix. FAK controls a number of adhesion-dependent cellular functions, including migration, proliferation and survival. Although FAK is overexpressed and activated in metastatic tumours, where it promotes invasion, it can also be elevated in cancers that have yet to become invasive. The contribution of FAK to the early stages of tumourigenesis is not known. We have examined the effect of activating FAK in non-transformed mammary epithelial cells (MECs) to understand its role in tumour initiation. In agreement with previous studies, we find FAK activation in 2D-culture promotes proliferation, migration, and epithelial-to-mesenchymal transition. However in 3D-cultures that better resemble normal tissue morphology, mammary cells largely respond to FAK activation via suppression of apoptosis, promoting aberrant acinar morphogenesis. This is an acquired function of FAK, because endogenous FAK signalling is not required for normal morphogenesis in 3D-culture or in vivo. Thus, FAK activation may facilitate tumour initiation by causing resistance to apoptosis. We suggest that aberrant FAK activation in breast epithelia is dependent upon the tissue context in which it occurs.

Cell division is a period of danger for cells, as inaccurate segregation of chromosomes can lead to loss of cell viability or aneuploidy. In order to protect against these dangers, cells ultimately initiate mitochondrial apoptosis if they are unable to correctly exit mitosis. A number of important chemotherapeutics exploit this response to delayed mitotic exit, but despite this, the molecular mechanism of the apoptotic timer in mitosis has proved elusive. Some recent studies have now shed light on this, showing how passage through the cell cycle fine-tunes a cell's apoptotic sensitivity such that it can respond appropriately when errors arise.

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated. To investigate the function of Axl receptor tyrosine kinase in CKD we performed a sub-total nephrectomy and fed high phosphate (1%) diet to Axl+/+ and Axl-/- mice. Plasma Gas6 (Axl' ligand), renal Axl expression and downstream Akt signalling were all significantly up-regulated in Axl+/+ mice following renal mass reduction and high phosphate diet, compared to age-matched controls. Axl-/- mice had significantly enhanced uraemia, reduced bodyweight and significantly reduced survival following sub-total nephrectomy and high phosphate diet compared to Axl+/+ mice; only 45% of Axl-/- mice survived to 14 weeks post-surgery compared to 87% of Axl+/+ mice. Histological analysis of kidney remnants revealed no effect of loss of Axl on glomerular hypertrophy, calcification or renal sclerosis but identified significantly increased tubulo-interstitial apoptosis in Axl-/- mice. Vascular calcification was not induced in Axl+/+ or Axl-/- mice in the time frame we were able to examine. In conclusion, we identify the up-regulation of Gas6/Axl signalling as a protective mechanism which reduces tubulo-interstitial apoptosis and slows progression to end-stage renal failure in the murine nephrectomy and high phosphate diet model of CKD.

Mitosis is a moment of exquisite vulnerability for a metazoan cell. Failure to complete mitosis accurately can lead to aneuploidy and cancer initiation. Therefore, if the exit from mitosis is delayed, normal cells are usually removed by apoptosis. However, how failure to complete mitosis activates apoptosis is still unclear. Here, we demonstrate that a phosphorylated form of the BH3-only protein Bid regulates apoptosis if mitotic exit is delayed. Bid is phosphorylated on serine 66 as cells enter mitosis, and this phosphorylation is lost during the metaphase-to-anaphase transition. Cells expressing a nonphosphorylatable version of Bid or a BH3-domain mutant were resistant to mitotic-arrest-induced apoptosis. Thus, we show that Bid phosphorylation primes cells to undergo mitochondrial apoptosis if mitotic exit is delayed. Avoidance of this mechanism may explain the selective pressure for cancer cells to undergo mitotic slippage., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer is a disease in which normal physiological processes are imbalanced, leading to tumour formation, metastasis and eventually death. Recent biological advances have led to the advent of targeted therapies to complement traditional chemotherapy and radiotherapy. However, a major problem still facing modern medicine is resistance to therapies, whether targeted or traditional. Therefore, to increase the survival rates of cancer patients, it is critical that we continue to identify molecular targets for therapeutic intervention. The Inhibitor of Apoptosis (IAP) proteins act downstream of a broad range of stimuli, such as cytokines and extracellular matrix interactions, to regulate cell survival, proliferation and migration. These processes are dysregulated during tumourigenesis and are critical to the metastatic spread of the disease. IAPs are commonly upregulated in cancer and have therefore become the focus of much research as both biomarkers and therapeutic targets. Here we discuss the roles that IAPs may play in cancer, and the potential benefits and pitfalls that targeting IAPs could have in the clinic.

The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax's dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

Most cells undergo apoptosis through the intrinsic pathway. This is dependent on mitochondrial outer membrane permeabilisation (MOMP), which is mediated by the pro-apoptotic Bcl-2 family proteins, Bax and Bak. During apoptosis, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM), wherein it contributes to the formation of pores to release cytochrome-c. However, it remains unclear whether Bax translocation is sufficient to bring about MOMP or whether Bax requires further signals on the OMM to be fully activated. We have previously shown that during mammary epithelial cell anoikis, Bax translocation does not commit cells to MOMP and detached cells are rescued if survival signals from the extracellular matrix (ECM) are restored. These findings implied that a second signal is required for mitochondrial Bax to fully activate and cause MOMP. We now identify p38MAPK (mitogen-activated protein kinase) as this necessary signal to activate Bax after its translocation to mitochondria. The inhibition of p38MAPK did not prevent Bax translocation, but its activity was required for mitochondrial Bax to bring about MOMP. p38MAPK was activated and recruited to a high molecular weight mitochondrial complex after loss of ECM attachment. Artificially targeting p38MAPK to the OMM increased the kinetics of anoikis, supporting a requirement for its mitochondrial localisation to regulate Bax activation and drive commitment to apoptosis.

A fundamental aspect in metazoans is the ability of a cell to recognise its positional context within a tissue. This is important in both development and homeostasis, where cell proliferation, differentiation and apoptosis are strictly controlled to form and maintain tissues. Much information has been generated on how cells receive and interpret adhesion-mediated signals. The non-receptor tyrosine kinase, Fak (focal adhesion kinase) has received much attention with regard to adhesion mediated signalling, including its role in survival. Survival signals are required to suppress the default pathway of apoptosis. The ultimate outcome of apoptotic signalling is the release of factors from the mitochondria into the cytosol. How the defined signalling pathways that control apoptosis converge on the mitochondria is an area with many unresolved questions.

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo.

The Notch pathway is aberrantly activated in a wide range of cancers, including breast carcinoma, and is required to maintain the transformed phenotype of many of these tumors. Notch signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear how Notch activation can lead to a general suppression of apoptosis. We show here that Notch signaling induced an autocrine signaling loop that activates Akt in breast epithelial cells. This activation of Akt was necessary for Notch-induced protection against apoptosis in the nontransformed breast epithelial cell line MCF10A. Moreover, inhibiting Notch signaling in breast cancer cells induced a decrease in Akt activity and an increase in sensitivity to apoptosis. Finally, the inhibition of ASK1 by Akt was responsible for the protection from apoptosis induced by DNA damage, as it prevented c-Jun NH(2)-terminal kinase-mediated phosphorylation and activation of p53.

Different cell types interpret their distinct extracellular matrix (ECM) environments to bring about specific cell fate decisions, and can differentiate or undergo apoptosis depending on their local adhesive interactions. Apoptosis in response to an inappropriate ECM environment is termed ;anoikis', or homelessness. Several studies, utilising a variety of cell types, have indicated a common, crucial role for focal adhesion kinase (FAK) in suppressing anoikis. A wide range of different integrins can activate FAK, raising the question of how cell type specific effects are regulated. In this study, we have used a constitutively active form of FAK to examine the mechanism of FAK-mediated survival signalling in cell types from distinct embryonic lineages that show differing sensitivities to anoikis. We demonstrate that both fibroblasts and epithelial cells prevent anoikis through FAK activation. We show that FAK activates multiple downstream pathways in order to suppress anoikis. However FAK regulates survival through a more restricted set of pathways in the more anoikis-sensitive epithelial cells. Furthermore, we identify a novel role for paxillin in apoptosis suppression.

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

Most defective and unwanted cells die by apoptosis, an exquisitely controlled genetic programme for removing such cells without damaging the surrounding tissue. Once a cell has committed to apoptosis, the process is remarkably efficient, and is completed within a few minutes of initiation. This point of no return for an apoptotic cell is commonly held to be the point at which the outer mitochondrial membrane is permeabilised, a process regulated by the Bcl-2 family of proteins. How these proteins regulate this decision point is central to diseases such as cancer where apoptotic control is lost. In this review, we will discuss apoptotic signalling and how a cell makes the irreversible decision to die. We will focus on one set of survival signals, those derived by cell adhesion to the extracellular matrix (ECM), and use these to highlight the complexities of apoptotic signalling. In particular, we will illustrate how multiple signalling pathways converge to determine critical cell fate decisions.

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.

Anoikis is a rapid apoptosis response that is initiated within a few minutes after inhibition of integrin signaling. In mammary epithelia, anoikis is mediated by subcellular translocation of Bax from the cytosol to mitochondria where it activates the intrinsic apoptosis pathway. The Bcl-2 homology 3 domain-only protein, Bim, has been proposed to have a key role in the apoptosis response of an epithelial cell line with reduced sensitivity to loss of integrin signaling, which undergoes apoptosis over a period of several days in suspension culture. Here we tested the involvement of Bim in the rapid anoikis response of mouse mammary epithelial cells and discovered that Bim does not have a role in detecting integrin-mediated signals. Instead Bim senses the loss of survival cues mediated by epidermal growth factor. Cell lines selected over many passages in culture have lost much of their sensitivity to anoikis signals arising from an altered cellular microenvironment and may undergo apoptosis through acquired mechanisms.

Epithelial cells require adhesion to the extracellular matrix for survival, and in the absence of adhesion they undergo apoptosis (anoikis). This is distinct from apoptosis induced by extracellular death ligands, such as tumor necrosis factor, which result in direct activation of caspase 8. Bid is a member of the BH3-only subfamily of the Bcl-2 proteins and is important for most cell types to apoptose in response to Fas and tumor necrosis factor receptor activation. Caspase 8 cleaves full-length Bid, resulting in truncated p15 tBid. p15 tBid is potently apoptotic and activates the multidomain Bcl-2 protein, Bax, resulting in release of cytochrome c from mitochondria. We have previously shown that Bax rapidly translocates from the cytosol to mitochondria following loss of adhesion and that this is required for anoikis. We have now examined the role of Bid in anoikis. Bid translocates to mitochondria with identical kinetics as Bax. Although Bid is required for anoikis, it does not require proteolytic cleavage by caspase 8. Furthermore, it does not require Bid to interact directly with other Bcl-2 family proteins, such as Bax. Our data indicate that Bid is important for regulating apoptosis via the intrinsic pathway and has implications for how Bid may fulfill that role.

Anoikis is apoptosis induced by loss of cell adhesion or inappropriate cell adhesion. Adhesion on the extracellular matrix is important to determine whether a cell is in the correct location and to delete displaced cells by apoptosis. The ability to overcome this requirement has important implications for metastatic cancer. However, how adhesion signals are interpreted by a cell into a life or death decision is complex. In this paper, we will examine this from the point of view of the apoptotic machinery of the cell, and discuss the various ways in which adhesion can influence this process., (Copyright 2004 Biochemical Society)

The function of exocrine glands depends on signals within the extracellular environment. In the mammary gland, integrin-mediated adhesion to the extracellular matrix protein laminin co-operates with soluble factors such as prolactin to regulate tissue-specific gene expression. The mechanism of matrix and prolactin crosstalk and the activation of downstream signals are not fully understood. Because integrins organize the cytoskeleton, we analysed the contribution of the cytoskeleton to prolactin receptor activation and the resultant stimulation of milk protein gene expression. We show that the proximal signalling events initiated by prolactin (i.e. tyrosine phosphorylation of receptor and the associated kinase Jak2) do not depend on an intact actin cytoskeleton. However, actin networks and microtubules are both necessary for continued mammary cell differentiation, because cytoskeletal integrity is required to transduce the signals between prolactin receptor and Stat5, a transcription factor necessary for milk protein gene transcription. The two different cytoskeletal scaffolds regulate prolactin signalling through separate mechanisms that are specific to cellular differentiation but do not affect the general profile of protein synthesis.

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.

The proapoptotic activity of the "BH3-only" molecule BAD can be differentially regulated by survival factor signaling. Bad-deficient mice lacking both BAD long and BAD short proteins proved viable, and most cell types appeared to develop normally. BAD did not exclusively account for cell death after withdrawal of survival factors, but it was an intermediate for epidermal growth factor- or insulin-like growth factor I-countered apoptosis, consistent with a "sensitizing" BH3-only molecule. Lymphocytes developed normally with no premalignant hyperplasia, but they displayed subtle abnormalities in proliferation and IgG production. Despite the minimal phenotype, Bad-deficient mice progressed, with aging, to diffuse large B cell lymphoma of germinal center origin. Exposure of Bad-null mice to sublethal gamma-irradiation resulted in an increased incidence of pre-T cell and pro-/pre-B cell lymphoblastic leukemia/lymphoma. Thus, proapoptotic BAD suppresses tumorigenesis in the lymphocyte lineage.

Adhesion of many cell types to the extracellular matrix is essential to maintain their survival. In the absence of integrin-mediated signals, normal epithelial cells undergo a form of apoptosis termed anoikis. It has been proposed that the activation of initiator caspases is an early event in anoikis, resulting in Bid cleavage and cytochrome c release from mitochondria. We have previously demonstrated that the loss of integrin signaling in mammary epithelial cells results in apoptosis and that this is dependent upon translocation of Bax from the cytosol to the mitochondria. In this paper, we ask whether caspases are required for Bax activation and the associated changes within mitochondria. We show that Bax activation occurs extremely rapidly, within 15 min after loss of integrin-mediated adhesion to extracellular matrix. The conformational changes associated with Bax activation are independent of caspases including the initiator caspase-8. We also examined downstream events in the apoptosis program and found that cytochrome c release occurs after a delay of at least 1 h, with subsequent activation of the effector caspase-3. This delay is not due to a requirement for new protein synthesis, since cycloheximide has no effect on the kinetics of Bax activation, cytochrome c release, caspase-3 cleavage, or apoptosis. Together, our data indicate that the cellular decision for anoikis in mammary epithelial cells occurs in the absence of caspase activation. Moreover, although the conformational changes in Bax are rapid and synchronous, the subsequent events occur stochastically and with considerable delays.

The explosion of signal transduction research over the last 10 years has provided a unique insight into the complexity of these intricate pathways. Whereas intermediates of multiple signaling pathways have been identified, understanding their function and, in particular, the interactions between them has become a daunting task. The increasing evidence that many of these pathways can cross-talk with each other via signal transactivation inevitably raises the question of how cells determine specificity in signaling. Despite the mind-numbing complexity of these pathways, progress has been made in developing highly specific and potent signal transduction inhibitors (STIs). STIs show promise in the treatment of cancer in preclinical studies and are currently in a number of clinical trials. Whereas many of these agents were "rationally designed," we barely understand their mechanisms of action. This review will highlight how recent studies using these STIs have elucidated novel mechanisms of STI action that may be used in the development of new therapeutic strategies for the treatment of cancer.

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.

Apoptosis is of important significance in the pathogenesis of cancer. Many methods are available for the measurement of apoptosis but the 'gold standard' is to identify apoptotic cells by their morphological features using microscopy. Caspase 3 is a cytosolic enzyme that is activated only in cells committed to undergo apoptosis. The activation of caspase 3 precedes the development of the classical morphological features of apoptosis. Using immunohistochemistry with an antibody against the active form of caspase 3, the apoptotic index (AI) was measured in 116 samples of human breast tissue (22 normal/benign and 94 invasive carcinomas). The AI obtained by measuring caspase activation has a strong correlation with the AI derived by morphological assessment (r = 0.736, P < 0.01). The AI is higher in the invasive group than in the benign group (P = 0.008), and in invasive cancer high AI is associated with high tumour grade (P = 0.013), positive node status (P < 0.001) and negative steroid receptor status (P = 0.001 for ER; P = 0.004 for PR). No significant association is observed between AI and tumour size. Measurement of apoptosis by immunohistochemistry using an antibody against the active form of caspase 3 is therefore reliable and correlates strongly with morphological assessment.

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax.

Tissue architecture in multicellular organisms is maintained through adhesive interactions between cells and their neighbors, and between cells and the underlying extracellular matrix. These interactions are important in the dynamic regulation of tissue organization as well as the control of cell proliferation, differentiation and apoptosis. The ultimate goal of this regulation is to promote cell growth and differentiation only when the cell is in the correct location, and to delete cells that have become displaced from their proper environment. It therefore plays an important role in development and tissue remodeling. In this review we consider the molecular mechanisms by which cell-matrix interactions contribute to cell survival, and discuss their role in mammary gland development and function.

Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (PTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.